In 1989, we reported that a nucleic acid-resistant phosphorothioate oligodeoxynucleotide with an antisense construct against the rev gene (alpha-rev) was active in suppressing the expression of HIV-1(IIIB) in chronically infected H9 cells (Matsukura et al., Proc. Natl. Acad. Sci. USA., 86:4244-4248, 1989). In the present study, we asked whether this alpha-rev could exert comparable antiviral activity in a variety of chronically infected target cell lines using p24 Gag protein production as an endpoint. When H9 cells were stably infected with 9 different HIV-1-strains (3 laboratory strains: HIV-1(IIIB), HIV-1MN, HIV-1RF; 6 clinical isolates: HIV-1ERS(101), HIV-1ERS(201pre), HIV-1ERS(201post), HIV-1ERS(206), HIV-1(018pre), HIV-1(018post), alpha-rev could provide a substantial suppression (equal to or greater than 60% suppression) in the expression of only three of the virus strains, HIV-1(IIIB), HIV- 1(201pre), and HIV-1(018post). When 6 different cell lines (H9, CEM, HUT102, MOLT4, U937, and G11) were stably infected with HIV-1(IIIB), HIV-1(IIIB) was sensitive to alpha-rev suppression only when harbored in H9 cells and CEM cells. The uptake and intracellular localization of fluorescein-labeled alpha-rev in each cell line did not correlate with the sensitivity of HIV-1 to the oligomer. The rates by which alpha-rev degraded intracellularly in each cell line also did not show detectable correlation. Levels of rev transcripts expressed in each cell line did not appear to correlate with the susceptibility of the virus to alpha-rev.